Because of the recent concern voiced about the accumulation of phthalate esters in the environment, we propose to investigate the metabolism of the phthalic acids and their esters by soil and water bacteria. The immediate objectives are: (a) to establish the catabolic routes used by bacteria to degrade those phthalates, (b) to study the hydroxylase(s) involved in the transformation of o-phthalate to 4,5-dihydroxyphthalate and characterize the initial intermediate(s) i.e., to determine whether a dihydrodiol or a monohydroxy derivative is formed first, (c) to purify and characterize 4,5-dihydroxyphthalate decarboxylase, the non-oxidative decarboxylative /// in the o-phthalate pathway of Ps. putida, and (d) to provide information on the biodegradability of the phthalate esters that are commonly used and have appeared as pollutants of the environment; di-2-ethylhexylphthalate and di-n-butylphthalate will be used as examples since these are the most abundant. The information obtained from these four specific objectives will be related to the well established catabolic sequences used by bacteria for the degradation of aromatic compounds. Of particular interest will be the specificity of the hydroxylases and decarboxylases, the initial products of the hydroxylation reactions, since cis-dihydrodiols have only been described as intermediates of aromatics with non-polar substituents, and the frequency and mechanism of non-oxidative decarboxylation reactions for degradative sequences. The metabolic pathways will be determined by the accumulation and identification of intermediates by whole cell and extracts; the respiratory patterns of induced and non-induced cells; isolation of mutants blocked in the pathways to prepare early intermediates. Phthalate hydroxylase(s) and 4,5-dihydroxyphthalate decarboxylase will be purified by standard chromatographic procedures and the proteins examined for subunit structure, cofactor requirements, catalytic activities and specificity. The biodegradability of the phthalate esters will be examined with enrichment cultures and perfusion systems, and the esterase activities of these, and pure typed cultures will be analyzed for phthalate ester hydrolysis.